MeDIP Real-Time qPCR has the Potential for Noninvasive Prenatal Screening of Fetal Trisomy 21.

This study aimed to verify the reliability of the 7 tissue differentially methylated regions used in the methylated DNA immunoprecipitation (MeDIP) real- time quantitative polymerase chain reaction (real-time qPCR) based approach of fetal DNA in maternal blood to diagnosis of fetal trisomy 21. Forty pregnant women with high risk pregnancy who were referred after first or second trimester screening tests, were selected randomly. For each sample whole DNA extraction (mother and fetus), fragmentation of DNA, immunoprecipitation of methylated DNA and real- time qPCR using 7 primer pairs was performed. D-value for each sample was calculated using the following formula D = -4.908+ 0.254 XEP1+ 0.409 XEP4+ 0.793 XEP5+ 0.324 XEP6+ 0.505 XEP7+ 0.508 XEP9+ 0.691 XEP12. In all normal cases, D value was negative, while it was positive in all trisomy cases. Therefore, all normal and trisomy 21 cases were classified correctly which correspond to 100% specificity and 100% sensitivity for this method. The MeDIP real-time qPCR method has provided the opportunity for noninvasive prenatal diagnosis of fetal trisomy 21 to be potentially employed into the routine practice of diagnostic laboratories.

many other health problems (1,2,6,7). While most fetal aneuplordy leads to miscarriage, trisomy 21 has the maximum survival rate. Due to the highest survival rate, the prenatal detection of fetal trisomy 21 is one of the commonest reasons for referral of women for prenatal diagnosis (8,9). The incidence of births of children with DS rises with the age of the mother. Screening for DS is an important part of routine prenatal care. Prenatal diagnosis was presented in the 1970s with the major aim of detecting common aneuploidies such as trisomies 21, 18 and 13 (7,10). Screening tests like the first trimester combination test are nowadays accessible to all pregnant women. Risk calculation is based on maternal age, nuchal translucency (NT) measurement by sonography and two serum markers: free beta hCG (free β-hCG) and pregnancy-associated plasma protein-A (PAPP-A).
The test properties are rather good with a detection rate of almost 85-90% with a false-positive rate of 5-9% (9,11).
Following a positive prenatal screening test, women are usually recommended to perform fetal karyotyping, which is considered as the gold standard to confirm the presence or absence of aneuploidies. Despite that, the main problem of karyotyping is the long period of time needed to achieve definitive results. Other faster and cheaper methods which have been introduced include interphase FISH and QF-PCR, but their main disadvantage is that they do not provide a full graphic demonstration of all chromosomes (12)(13)(14)(15).
Prenatal genetic diagnosis of DS and other aneuploidies is done using common cytogenetic tests or DNA analysis which needs fetal DNA to be obtained by invasive methods such as chorionic villus sampling (CVS) during the first trimester and amniocentesis during the second trimester (16)(17)(18).
Even so, all these methods are invasive and associated with risk of fetal loss (12,19,20).
Over the last few years, a great quantity of investigation have been accomplished on the development of noninvasive prenatal testing (NIPT) for fetal aneuploidies (23,24). The discovery of cell free fetal DNA (cffDNA) in the maternal circulation has opened up a new horizon in the field of prenatal care and screening. Detection of chromosomal aneuploidy is a challenging goal in NIPD research (22,(25)(26)(27) (20,(28)(29)(30)(31). DNA methylation is a dynamic process and could change during development. It is believed that more than half of tissue differentially methylated regions (TDMRs) are methylated in embryonic tissues and during the differentiation, they undergo de-methylation process.These TDMRs have been used to enrich and assess fetal DNA ratio by using monoclonal antibody for methylated CpGs using MeDIP approach (16,18,32,33).
The aim of this study was to evaluate and validate the methylated DNA immunoprecipitation of fetal DNA in maternal blood for diagnosis of fetal trisomy 21.

Sample collection and processing
The samples included 40 pregnant women referred between October 2014 to December 2015 to the Medical Genetic Center of Genome (Iran, Isfahan). All pregnant women agreed to participate in the study and signed an informed consent. The study was approved by the Bioethics Committee of Isfahan University of Medical Sciences. The experimental procedure was followed as previously described with some modifications (16,33). The participants were women with singleton pregnancies, between 13 and 21 weeks of gestation. All participants underwent invasive prenatal diagnosis by CVS or amniocentesis followed by FISH or  (33). The real-time PCR was performed with specific primers (Table 1) and  Finally, D value amount for each unknown sample was calculated using the following formula (33): Where IP correspond to Immunoprecipitated and PB represent the peripheral blood. where X EPi is fraction value for each EP marker (33).

Results
The ratio values obtained from the 7 selected DMRs (    expensive. Moreover, this method will be able to offer results in less than 3 working days (16).
As different ethnic groups may have different DNA methylation patterns and this could influence MeDIP-qPCR results, the main purpose of the present research was to evaluate and assess an optimized condition for NIPD of fetal trisomy 21 using cffDNA in maternal blood in Iran. We found that our results were in accordance with previous studies and this method is usable for screening in Iran. Furthermore, many different studies will need to be implemented to support the introduction of a new diagnostic strategy into the clinical practice of prenatal diagnostic laboratories.
This approach has provided the opportunity for NIPD of fetal trisomy 21 into many diagnostic laboratories (19,35). Although the present study is based on a small sample of participants and data from more samples will be of help, our results confirm that this technology could be effective for screening trisomy 21 in pregnant women and could be applied in clinical practice.